Silks are protein fibres produced by a wide range of insect and spider species. The silk of the domesticated silkworm, Bombyx mori, has been used as a suture biomaterial for centuries. Numerous efforts to clone and express silkworm or spider silks in transgenic systems have found it a Herculean task. The large sizes and highly repetitive sequences of these silk genes make them recalcitrant to expression outside specialized silk glands, and lead to low protein yields.
Although silkworm cocoons and spider webs are the best known silks, other species may produce silks better suited to transgenic production. Honeybee larvae (Apis mellifera) spin silk cocoons in which they pupate. Honeybee silk is encoded by four small (˜30 kDa each) and non-repetitive fibre genes (Sutherland et al., 2006). Homologous sets of four genes have also been found in bumblebees, bulldog ants, weaver ants, hornets and Asiatic honeybees (Sutherland et al., 2007; Sezutsu et al., 2007; Shi et al., 2008; WO 2007/038837).
Vintage x-ray fibre diffraction work demonstrated that honeybee silk contains α-helical proteins assembled into a coiled-coil conformation, most likely a tetrameric coiled-coil structure (Atkins, 1967), with the four strands likely corresponding to the four different silk proteins. Bioinformatics techniques predict that each of the honeybee silk protein sequences contains 60-68% coiled-coil (Sutherland et al., 2006).
Silk threads can be hand-drawn from the silk glands of honeybee larvae. These threads are less strong but more extensible and tougher than silkworm silk fibres (Hepburn et al., 1979).
Shi et al. (2008) recently reported recombinant production of Asiatic honeybee silk (Apis cerana). The four A. cerana silk proteins were expressed in a soluble form in Escherichia coli with yields of 10-60 mg per liter of ferment. A variety of experimental techniques were used to characterize the structure and interactions of the proteins at low concentration (0.03 to 0.2 wt %). These conclusively demonstrated that neither the individual proteins nor a mix of four proteins had tight tertiary packing in solution. The proteins existed as monomers or loosely associated dimers and had predominantly random-coil conformation with little α-helical structure.
There is a need for further methods to produce silk dope from recombinantly expressed coiled-coil silk proteins which can be used to manufacture a wide variety of products.